The 5-substituted hydantoin racemase enzyme (hereinafter abbreviated to HRase) catalyzes a racemization reaction of an optically active 5-substituted hydantoin compound, i.e., a D- or L-5-substituted hydantoin compound.
The 5-substituted hydantoin compound undergoes a hydrolysis reaction with hydantoinase (1) and Carbamyl amino acid hydrolase (2) to form an amino acid as shown in the following reaction scheme (I):

As shown in the reaction scheme above, the hydantoinase hydrolyzes the 5-substituted hydantoin compound thereby forming N-carbamyl amino acid. Additionally, the N-carbamyl amino acid hydrolase hydrolyzes N-carbamyl amino acid thereby forming an optically active amino acid. These enzymes should be optically selective.
Methods of using a microbial enzyme system and combining a bacterial enzyme system with a chemical reaction system have been previously described. Production of optically amino acids from 5-substituted hydantoin compounds is important in the production of pharmaceutical preparations, chemical industry products, food additives and other similar articles or products.
The optico-selective hydrolysis should enable the efficient racemization of one enantiomer to another enantiomer, the second enantiomer can further serve as a substrate, for example see the above reaction scheme. Furthermore, this conversion can be performed using microbial enzymes or microbial enzymes combined with a chemical reaction system. Under neutral conditions where the enzyme system is active, the racemization of the optically active 5-substituted hydantoin compound, not serving as the substrate, is very low. Therefore, the racemization becomes rate-determining and resulting in poor conversion into the optically active amino acid.
Accordingly, for the purpose of racemization of the optically active 5-substituted hydantoin compound under neutral conditions, HRase was searched for, and HRases derived from microorganisms of the genus Arthrobacter (Japanese Patent Application Laid-Open (JP-A) No. 62-122591; Ann. N.Y. Acad. Sci., 672, 478; Japanese Patent Application Laid-Open (JP-A) No. 6-343462) and microorganisms of the genus Pseudomonas (Japanese Patent Application Laid-Open (JP-A) No. 4-271784; J. Bacteriol., 174, 7989 (1992)) have been reported. The optimum reaction temperatures of the previously reported HRases derived from microorganisms are 37° C. for the enzyme derived from Arthrobacter sp. DSM-3747 (Ann. N.Y. Acad. Sci., 672, 478), 10 to 50° C. for the enzyme derived from Arthrobacter sp. DK200 (Japanese Patent Application Laid-Open (JP-A) No. 62-122591), and 45° C. for the enzyme derived from Pseudomonas sp. NS671.
Generally, when the working optimum temperature of an enzyme is increased, the industrial utility value of the enzyme is also increased. That is, if the reaction temperature can be increased, the reaction rate can be also increased, which results not only in efficient progress of the desired reaction but also in a reduction in the risk of contamination of the reaction solution with microorganisms during the reaction. This results in several advantages including easy process and quality control.
As described above, HRase having a higher optimum temperature for the reaction is useful from the viewpoint of industrial applicability. However, the upper limit of the working optimum temperatures of the previously reported HRases is 50° C. and an HRase having a higher working optimum temperature is desired to achieve efficient progress of the racemization reaction and to reduce the risk of contamination of the reaction solution with microorganisms.